In diseases of non-cardiogenic pulmonary edema, endothelium becomes disrupted allowing passage of macromolecules into the extravascular space. Endothelial disruption in thee diseases is a regulated process that is likely initiated by release of toxic oxygen radicals and proteases by activated which cells, ischemia-reperfusion, and/or neurohumoral inflammatory and vasoactive factors. These stimuli elevate endothelial cell intracellular free Ca2+ ([Ca2=]I) which directly or indirectly reorganizes the cytoskeleton and promotes myosin light chain phosphorylation leading to actinomyosin interaction and contraction. Cytoskeletal reorganization and contraction together cause generation of intercellular gaps and permeability. While elevated [Ca2+]I promotes permeability, elevated cAMP opposes permeability. We therefore investigated the relationship between [Ca2=]I cAMP content in pulmonary microvascular endothelial cells. Expression of multiple isoforms of adenylyl cyclase were identified, including on Ca2+ inhibitable isoform; however, the activity of a Ca2+ inhibitable adenylyl cyclase predominated in intact cells and established an inverse relationship between [Ca2+]I and cAMP in permeability induced by [Ca2+]I agonists. This proposal tests the overall HYPOTHESIS that Ca2+ inhibition of cAMP formation, which is established by a Ca2+ inhibitable adenylyl cyclase, is an important amplification step in the development of endothelia permeability. The hypothesis will be tested using inflammatory mediators thrombin and thapsigargin, which elevate [Ca2+]I via different mechanisms. The SPECIFIC AIMS are to test the HYPOTHESES that: [1] ca2+ inhibitable adenylyl cyclase establishes an inverse relationship between [Ca2+]I and cAMP content in pulmonary microvascular endothelium; and, [2] Ca2+ inhibitable adenylyl cyclase promotes filamentous-actin reorganization, myosin light chain phosphorylation, intercellular gap formation, and permeability in pulmonary endothelium (macro and microvascular). It is hoped completion of these studies will improve our understanding of mechanisms which regulate endothelial barrier properties so that effective therapies can be developed for treatment of non-cardiogenic pulmonary edema.